J Mol Biol. Author manuscript; available in PMC 2009 April 3.
Published in final edited form as:
J Mol Biol. 2008 August 1; 381(1): 174-188.
Published online 2008 June 7. doi:  10.1016/j.jmb.2008.05.084
PMCID: PMC2665032
NIHMSID: NIHMS97250
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Structural Basis of Transcriptional Regulation of the Proline
Utilization Regulon by Multifunctional PutA
Yuzhen Zhou,1† John D. Larson,2† Christopher A. Bottoms,3 Emilia
C. Arturo,2 Michael T. Henzl,4 Jermaine L. Jenkins,4 Jay C. Nix,5
Donald F. Becker,1* and John J. Tanner2,4*
1 Department of Biochemistry, University of Nebraska-Lincoln, Lincoln,
NE 68588, USA
2 Department of Chemistry, University of Missouri-Columbia, Columbia,
MO 65211, USA
3 Department of Computer Science, University of Missouri-Columbia,
Columbia, MO 65211, USA
4 Department of Biochemistry, University of Missouri-Columbia,
Columbia, MO 65211, USA
5 Molecular Biology Consortium, Lawrence Berkeley National Laboratory,
Berkeley, CA 94720
* Corresponding authors. E-mail addresses: dbecker3/at/unl.edu, Email:
tannerjj/at/missouri.edu
†These authors contributed equally to this research.
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Summary
The multifunctional Escherichia coli PutA flavoprotein functions as
both a membrane-associated proline catabolic enzyme and transcriptional
repressor of the proline utilization genes putA and putP. To better
understand the mechanism of transcriptional regulation by PutA, we have
mapped the put regulatory region, determined a crystal structure of the
PutA ribbon-helix-helix domain (PutA52) complexed with DNA and examined
the thermodynamics of DNA binding to PutA52. Five operator sites, each
containing the sequence motif 5′-GTTGCA-3′, were identified using
gel-shift analysis. Three of the sites are shown to be critical for
repression of putA, whereas the two other sites are important for
repression of putP. The 2.25 Å resolution crystal structure of PutA52
bound to one of the operators (operator 2, 21-bp) shows that the
protein contacts a 9-bp fragment, corresponding to the GTTGCA consensus
motif plus three flanking base pairs. Since the operator sequences
differ in flanking bases, the structure implies that PutA may have
different affinities for the five operators. This hypothesis was
explored using isothermal titration calorimetry. The binding of PutA52
to operator 2 is exothermic with an enthalpy of -1.8 kcal/mol and a
dissociation constant of 210 nM. Substitution of the flanking bases of
operator 4 into operator 2 results in an unfavorable enthalpy of 0.2
kcal/mol and 15-fold lower affinity, which shows that base pairs
outside of the consensus motif impact binding. The structural and
thermodynamic data suggest that hydrogen bonds between Lys9 and bases
adjacent to the GTTGCA motif contribute to transcriptional regulation
by fine-tuning the affinity of PutA for put control operators.
Keywords: proline utilization A, X-ray crystallography, isothermal
titration calorimetry, ribbon-helix-helix, proline catabolism
Introduction
Proline is used as a source of carbon, nitrogen and energy through two
oxidative steps catalyzed by proline dehydrogenase (PRODH) and
Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH).1-7 In
enteric bacteria such as Escherichia coli, proline utilization requires
the two genes, putP and putA. The former encodes the PutP high-affinity
Na+-proline transporter and the latter encodes the multifunctional
flavoprotein PutA (Proline utilization A).8,9 PutA is unique
in that it functions as both a transcriptional repressor of the put
genes and a membrane-associated bifunctional proline catabolic
enzyme.2,10-12 The enzymatic and transport functions
of the putA and putP genes, respectively, are conserved among different
Gram-negative bacteria, whereas the genetic organization and regulatory
mechanisms that control the expression of these genes are highly
divergent.7,10-18 The focus of this work is to provide
a molecular and structural understanding of the regulation of put genes
in E. coli by PutA.
PutA from E. coli combines PRODH, P5CDH, and transcriptional regulatory
activities into a single polypeptide of 1320 amino
acids.2,19 Insights into the organization of the functional
domains in PutA have been gained from molecular dissection and
characterization of truncated PutA proteins. The PRODH and P5CDH active
sites are located within residues 261-612 and 650-1130, respectively,
with the PRODH active site utilizing an FAD cofactor and P5CDH activity
requiring NAD+. Structural studies have shown that the PRODH domain
forms a unique (βα) barrel,20,21 and that reduction by
dithionite causes dramatic conformational changes in the FAD ribityl
chain.22 Molecular dissection studies showed that the DNA-binding
domain is contained in residues 1-47.23 Subsequently, the crystal
structure of a polypeptide corresponding to E. coli PutA residues 1-52
(PutA52) was solved, which showed that PutA is a member of the
ribbon-helix-helix (RHH) family of transcriptional
regulators.23,24
While knowledge of PutA structure and function continues to build, a
considerable gap remains in our understanding of critical PutA-DNA
interactions in the put control DNA region. To further understand the
regulation of proline metabolism in E. coli, we have identified the
PutA binding sites in the put regulatory region, elucidated the roles
of these operators in repressing expression of putA and putP,
determined the crystal structure of PutA52 bound to one of the
identified operators, and investigated the thermodynamics of DNA
binding to PutA52 using isothermal titration calorimetry.
Results
Identification of PutA binding sites
Initial localization of PutA binding sites in the put control DNA
region was performed by gel mobility shift assays using different
fragments of the 419-bp put control DNA. Systematic evaluation of
different regions of the put control DNA indicated that PutA does not
bind to the 1-170 bp region immediately downstream of putP (Fig.
1a, lanes 3-4). However, PutA was observed by gel mobility shift assays
to bind to regions 183-231 and 342-412 of the put control DNA (data not
shown). Additional assays indicated that PutA binds to oligonucleotides
183-210, 342-365 and 388-412 (Fig. 1a, lanes 5-10). Previously, we
showed by gel mobility shift assays that PutA also binds to
oligonucleotide 211-231, with apparent binding stoichiometry of one DNA
duplex per PutA dimer.25
Figure 1
Figure 1
Localization of PutA binding sites in the put control DNA region. (a)
Gel mobility shift assays of PutA with different regions of put control
DNA. Separate binding mixtures of PutA (0-1.5 μM) with full-length put
control DNA 1-419 (more ...)
Sequence alignment of the four oligonucleotides that bound to PutA
(183-210, 211-231, 342-365, 388-412) revealed a GTTGCA consensus
sequence. This motif is present in each of the four oligonucleotides
(Fig. 1b), and it appears five times in the 183 - 412 bp region of
the put control DNA (Fig. 1b). Thus, five potential operator sites,
denoted O1 - O5, were proposed, as shown in Fig. 1c.
The proposed binding sites were further examined by changing each one
from GTTGCA to GTCATA by site-directed mutagenesis of the put control
DNA. Gel mobility shift assays show that simultaneously mutating all
five sites disrupts PutA binding to the put control DNA (Fig. 2a,
Δ12345) confirming that PutA specifically recognizes only the five
binding sites in the put control region. Gel mobility shift assays were
then used to test PutA binding to the five sites incrementally using
PutA52 to resolve the different complexes. As shown in Fig. 2b,
decreasing the number of binding sites in the put control DNA reduces
the observed mobility shift of the protein-DNA complex. This further
confirms that the put control DNA contains five PutA binding sites, and
suggests that PutA52 is able to bind all five sites simultaneously
Figure 2
Figure 2
Gel mobility shift assays of full-length PutA and PutA52 with wild-type
put control DNA and put control DNA with an increasing number of
mutated binding sites. (a) Separate binding mixtures of full-length
PutA (0-0.25 μM) with wild-type (more ...)
Autorepression of putA
Cell-based reporter gene assays were performed to test the role of each
PutA binding site in repressing expression of putA. For these assays,
E. coli strain JT31 putA- lacZ- was cotransformed with PutA-pUC18 and
P[putA]:lacZ-pACYC184 constructs (wild-type and single or multiple
operator site mutations in the put control DNA). Western analysis
confirmed expression of PutA. Consistent with previous results, PutA
repressed expression of the lacZ reporter gene by over 75 % relative to
control cells (pUC18 alone and wild-type P[putA]:lacZ construct)
(Fig. 3a, WT).22 Mutations of O1 (Δ1) and O2 (ΔO2) singly or
in combination (ΔO1-2) did not increase β-galactosidase activity.
Because PutA repression of the lacZ reporter gene (~ 73 %) was not
diminished by mutating operator sites O1 and O2, PutA binding to these
sites is not necessary for repressing transcription of putA. Mutating
O3 (ΔO3) greatly reduced lacZ reporter gene expression in the control
cells (data not shown) to ~ 10 % of wild-type put control DNA. Because
of the intrinsically low reporter gene expression of the ΔO3 mutant
construct, we were not able to directly assess the impact of site O3 on
PutA repression of putA. O3 is located in the -35 region of the putA
promoter (see Fig. 1c), thus, the mutation at site O3 most likely
decreases the binding of the σ subunit of E. coli RNA polymerase to the
-35 element. We thus consider O3 to be an important operator for
autorepression of putA despite the fact that we could not test its role
using the reporter gene assay. Mutating sites O4 (ΔO4) or O5 (ΔO5)
increased β-galactosidase activity and lowered repression of the lacZ
reporter gene to about 50 % relative to the control cells (Fig.
3a). Simultaneously mutating O4 and O5 (ΔO4-5) generated an additive
effect with a 3-fold increase in β-galactosidase activity relative to
WT resulting in only 20 % repression of the lacZ reporter gene. Thus
O3, O4 and O5 are the most critical sites for PutA autorepression of
putA.
Figure 3
Figure 3
β-galactosidase activity from lacZ reporter constructs containing
various mutations in the put control DNA. (a) Relative percent
β-galactosidase activity in E. coli strain JT31 containing PutA-pUC18
and wild-type (WT) P[putA]:lacZ and various (more ...)
Regulation of putP by PutA
The binding sites critical for regulating putP were also identified. In
these assays, E. coli strain JT31 putA- lacZ- was cotransformed with
the PutA-pUC18 construct and the P[putP]:lacZ-pACYC184 construct
(wild-type and single or multiple operator site mutations in the put
control DNA). These results are shown in Fig. 3b. PutA repressed
lacZ reporter gene expression by about 47 % relative to control cells
(Fig. 3b, WT). Apparently PutA represses putP promoter activity
less than the putA promoter, consistent with previous results
suggesting PutA is a stronger regulator of putA than
putP.26,27 Mutation of O1 (ΔO1) increased β-galactosidase
activity thereby decreasing the repression of the lacZ reporter gene to
about 30 % relative to control cells. Mutating O2 singly (ΔO2) or in
combination with O1 (ΔO1-2) resulted in about 20 % repression relative
to control cells. In contrast to the putA promoter, mutation of O3, O4,
and O5 individually (ΔO3, ΔO4, ΔO5) (Fig. 3b) or in combination
(ΔO3-5) (data not shown) did not significantly increase β-galactosidase
activity or alter the repression of the lacZ reporter gene. Mutating
all five binding sites (ΔO1-5) resulted in the same repression of lacZ
expression (20 %) as ΔO1-2 put control DNA (Fig. 3b). Thus, PutA
binding to O1 and O2 is responsible for repressing the putP promoter.
Overall structure of PutA52 bound to O2
The crystal structure of PutA52 bound to O2 was solved in order to
understand the three-dimensional structural basis of DNA recognition by
PutA. This structure is the first one of a PutA RHH domain bound to
DNA, and it is currently the highest resolution structure of a RHH/DNA
complex. The asymmetric unit contains one PutA52 dimer bound to one O2
duplex (Fig. 4).
Figure 4
Figure 4
Overall structure of PutA52 bound to O2. PutA52 chains A and B are
colored green and magenta, respectively. DNA is represented as sticks,
with strand 1 colored yellow and strand 2 colored white. The electron
density map is a 2F[o]-F[c] map with F[c] and phases (more ...)
Each PutA52 chain adopts the RHH fold, which consists of a β-strand
(β1) followed by two α-helices (αA, αB). The two protein chains
assemble into a dimer featuring an intermolecular two-stranded
antiparallel β-sheet (Fig. 4).
The bound DNA ligand adopts the B conformation, based on analysis of
projected phosphorus positions (z[P]) using 3DNA.28 Values of z[P]
< 0.5 are diagnostic of B-form DNA, whereas z[P] > 1.5 Å indicate
A-form DNA.28,29 All but three of the 17 base pair steps of
O2 have z[P] < 0.5 Å. The three exceptions have z[P] = 0.52 - 0.58 Å.
Thus, binding of PutA52 to O2 does not cause significant distortion of
the DNA from the expected B conformation. Also, the double helix
displays no discernable curvature (Fig. 4).
The β-sheet of PutA52 inserts into the DNA major groove (Fig. 4).
Residues of the sheet contact DNA bases, while residues near the
N-terminus of αB interact with the DNA backbone. This general mode of
binding is typical for RHH proteins.30
Although the five operators that we identified each contain the 6-bp
consensus sequence of GTTGCA (Fig. 1b), the structure shows that
PutA52 contacts a larger fragment of DNA. A plot of the surface area
buried by nucleotides in the protein-DNA interface is shown in Fig.
5a. The bimodal shape of the plot reflects the two-fold symmetries of
the protein dimer and the DNA double helix. The surface area
calculations, along with detailed inspection of the protein-DNA
interface, show that the footprint of PutA52 encompasses the 9-bp
fragment from G6:C16 to C14:G8 (see boxed base pairs in Fig. 4).
Note that this fragment contains the GTTGCA motif. Interactions with
the 9-bp fragment are summarized schematically in Fig. 5b. and
shown in detail in Fig. 6.
Figure 5
Figure 5
Footprint of PutA52 on O2 derived from the crystal structure. (a)
Surface area contributed by DNA nucleotides to the protein-DNA
interface. (b) Schematic diagram of protein-DNA interactions. Dotted
lines indicate electrostatic interactions. Thick, solid (more ...)
Figure 6
Figure 6
Detailed stereographic view of the protein-DNA interface. PutA52 chains
A and B are colored green and magenta, respectively. DNA strands 1 and
2 are colored yellow and white, respectively. Cyan spheres represent
water molecules. Dotted lined indicate (more ...)
Interactions with DNA bases
Structures of RHH domains bound to DNA show that, typically, two polar
residues and one Arg/Lys from each β-strand form hydrogen bonds to DNA
bases. In PutA, this critical triad corresponds to Thr5, Gly7 and Lys9,
and all three residues interact with DNA bases. We note that these
residues are identically conserved among PutAs.24
Lys9 binds to the pair of guanine bases located at the 5′ ends of each
strand (Fig. 5b). Lys9 of chain A interacts with the guanine bases
of strand 2, while Lys9 of the B chain interacts with the guanine bases
of strand 1. The two sets of interactions are nearly identical
(Fig. 6), which is expected since they involve the palindromic
ends of the DNA fragment. Each Lys9 forms four hydrogen bonds, two with
each base of the guanine pair. These interactions are shown for Lys9(B)
in Fig. 7a. The hydrogen bond distances are 2.5 - 3.1 Å for the
inner base (G7 of strand1, G9 of strand 2) and 3.2 - 3.5 Å for the
outer base (G6 of strand 1, G8 of strand 2). We note that only guanine
has two appropriately placed hydrogen bond acceptors for interaction
with Lys9, so these interactions appear to enforce a preference for
binding a 9-bp fragment containing GG at the 5′ ends of both strands.
Figure 7
Figure 7
Close-up views of selected protein-DNA interactions. In all three
panels, PutA52 chains A and B are colored green and magenta,
respectively, DNA strands 1 and 2 are colored yellow and white,
respectively, and black dotted lines indicate electrostatic (more
...)
Thr5 forms hydrogen bonds with three different base pairs and both DNA
strands. In chain A, the hydroxyl of Thr5 donates a hydrogen bond to T8
of strand 1 (Fig. 7a), while the backbone carbonyl accepts a
hydrogen bond from C12 of strand 2 (Fig. 7b). Since the hydrogen
bond with T8 involves the palindromic GGT end of the DNA, one might
expect Thr5(B) to form an analogous interaction with T10 of strand 2.
Interestingly, Thr5(B) accepts a hydrogen bond from C11 of strand 1
(Fig. 7b) rather than hydrogen bonding with T10. The expected
two-fold symmetry is broken by a conformational change of Thr5(B). The
χ angle of Thr5(B) is +60°, whereas this angle is -60° for Thr5(A).
We note that Thr5 has χ = -60° in all chains of ligand-free PutA52
structures (PDB codes 2AY0, 2GPE). Thus, binding to DNA induced a
conformational change in Thr5(B), which introduces asymmetry in PutA52.
Gly7 helps confer sequence specificity despite lacking a side chain. In
chain A, Gly7 donates a hydrogen bond (2.9 Å) to the N7 atom of G11
(Fig. 7b). In chain B, Gly7 forms van der Waals interactions with
the C5 methyl of T9 (Fig. 7c). Note also the close contacts
between DNA bases and Thr5(A) in this region of the structure
(Fig. 7c). The tight packing of the T9:A13 base pair against Gly7
and Thr5 could contribute to sequence specificity.
Finally, there are no water molecules bridging the protein with DNA
bases. There is, however, one water molecule (Wat6) strategically
located in the protein-DNA interface on the pseudo two-fold axis that
relates the two chains (Fig. 6). It is equidistant from the two
Gly7 residues of the β-sheet, and forms hydrogen bonds with G10 of DNA
strand 1 and G11 of strand 2 (Fig. 7B). Wat6 appears to fill the
void created by the lack of a side chain at residue 7. Indeed, mutation
of Gly7 in silico to any other residue causes steric clash with this
water molecule as well as with DNA bases.
Interactions with the DNA backbone
Thr28, Pro29 and His30 bind the DNA backbone. Thr28 is the N[cap] of
αB, while Pro29 and His30 are the first two residues of αB. The
interactions display nearly perfect two-fold symmetry (Fig. 6), so
just one set of interactions will be described. The side chains of
Thr28 and His30 form electrostatic interactions with the phosphate
group connecting the two G nucleotides at the 5′ end of the 9-bp
fragment (Fig. 7a). In addition, the backbone of His30 donates a
hydrogen bond to the phosphate group of the T nucleotide at the 5′ end
of the 9-bp fragment (T8 of strand 1, T10 of strand 2, see Fig.
7a). Finally, the C[δ] atom of Pro29 forms close contacts (3.4 Å) with
oxygen atoms of the phosphate backbone (Fig. 7a).
Isothermal titration calorimetry
The binding of O2 to PutA52 at pH 8.0 was studied using ITC to gain
insights into the thermodynamic basis of DNA recognition. In Tris
buffer, the association reaction was evidently endothermic (Fig.
8a), whereas, in phosphate buffer at the same pH, the reaction was
weakly exothermic (Fig. 8b). Since the enthalpy of ionization of
Tris (11 kcal/mol) differs substantially from that of dihydrogen
phosphate (1 kcal/mol), these results suggest that the DNA-binding
event is coupled to the ionization reaction of the buffer at pH 8.0.
Moreover, the fact that the titration in Tris yielded the more
endothermic result implies proton uptake by the protein-DNA complex
during association.
Figure 8
Figure 8
ITC-based analysis of the interaction between PutA52 and the O2 and
O2bf4 duplexes. (a) Raw data for the titration of 12 μM PutA52 with
0.15 mM O2 (10 μL additions) in NaCl, Tris, pH 8.0. (b) Raw data for
the titration of 21 μM (more ...)
The data from the four titrations with O2 were fit simultaneously as
described in Materials and Methods to estimate the intrinsic binding
enthalpy, association equilibrium constant and number of protons
transferred (Fig. 8c). This analysis shows that the binding of O2
to PutA52 is intrinsically exothermic, with ΔH = -1.8 kcal/mol
(Table 2), and K = 4.8 × 106 M-1, which corresponds to K[d] =
210 nM (Table 2). The latter value agrees favorably with the
estimate from gel-shift analysis of K[d] < 200 nM for O2 binding to
full-length PutA.25 The estimated number of protons transferred
to the protein/DNA complex is 0.7.
Table 2
Table 2
Isothermal titration calorimetry data for DNA binding to PutA52 at 298K
A second set of titrations was performed using oligonucleotide O2fb4,
which is identical to O2 except that the bases flanking the GTTGCA
motif are those of O4. These measurements were performed to assess the
impact of bases outside of the consensus motif on affinity. As with O2,
the apparent enthalpy of binding of O2fb4 to PutA52 at pH 8.0 is
dependent on buffer choice. In Tris buffer, the association appears to
be strongly endothermic (Fig. 8d), but in phosphate buffer the
reaction is nearly isenthalpic (Fig. 8e).
Global analysis of the data from the two O2fb4 titrations (Fig.
8f) shows that binding of this oligonucleotide to PutA52 is marginally
endothermic, with intrinsic enthalpy change of only 0.18 kcal/mol
(Table 2). The association constant from global fitting is K = 3.2
× 105 M-1, which corresponds to K[d] = 3100 nM. As with the O2
titrations, there is an uptake of 0.7 protons during binding, which
suggests that the binding mechanisms of the two ligands are
qualitatively similar. Notice, however, that the association constant
for O2 is fifteen times higher than that of O2fb4. These results show
that bases outside of the consensus motif impact the affinity of
PutA52, and presumably PutA, for put control sites.
The binding of PutA52 is entropy-driven for both ligands. This result,
combined with the observation that the protein-DNA interface is nearly
devoid of bound water molecules, suggests that desolvation of
macromolecular surfaces is important for DNA binding. We note that the
free energy of the RHH protein MetJ binding to a metbox operator also
includes a substantial favorable entropic component at 25 °C,
particularly in the absence of the corepressor
S-adenosylmethionine.31
Discussion
Transcriptional regulation of the put regulon
Based on the arrangement of the five PutA-DNA binding sites, PutA most
likely represses the put genes by hindering the σ70-dependent binding
of E. coli RNA polymerase to the putA and putP promoter
regions.32 We did not find additional PutA consensus binding
sites in the coding regions of putA and putP, indicating that PutA
binds only to the put intergenic region. Previous reports suggested
that proline, via PutA, regulates expression of putA more tightly than
putP.26,27 Here we have shown that PutA is a stronger
repressor of the putA promoter than the putP promoter. Therefore, putP
expression appears to be regulated relatively weakly by PutA, which
would allow proline uptake under a variety of environmental conditions
leading to subsequent activation of putA expression. In addition to
proline-specific regulation by PutA, the put genes are also responsive
to global regulators. The cAMP receptor protein has been proposed to
function as an activator by increasing putA and putP promoter activity
in nutrient-poor environments.26
We evaluated put control DNA sequences from other bacteria in which
PutA contains the conserved RHH domain and is predicted to function as
an autogenous transcriptional repressor. The GTTGCA sequence was found
in every putA promoter region of the 39 genome sequences analyzed.
We also examined put control DNA sequences in bacteria that share the
same genetic organization of putA and putP as found in E. coli.
Fig. S1 (supplemental material) shows an alignment of put control
DNA sequences from E. coli, Shigella boydii, Salmonella typhimurium,
Klebsiella aerogenes, Yersinia pestis and Pseudomonas putida. The
length of the intergenic region ranges from 361 base pairs in P. putida
to 577 base pairs in Y. pestis. Each sequence has at least three exact
repeats of the GTTGCA motif. Operators O1, O3 and O4 are present in all
six sequences. O2 is present in all six organisms, except P. putida. O5
is the least conserved site. Y. pestis and P. putida have additional
exact repeats of the motif that do not align with the E. coli sites.
These results suggest that the GTTGCA motif is the fundamental
transcriptional control element of the PutA autogenous repression
system. Therefore, the biochemical and structural results reported here
for E. coli are likely applicable to other organisms in which PutA
serves as a transcriptional repressor.
Importance of the GTTGCA motif for PutA - operator binding
Mutation of the consensus GTTGCA motif to GTcatA was found to severely
impact binding to PutA, based on gel shift analysis. In fact, mutation
of all five operators eliminated binding. Moreover, this mutation was
found to affect gene transcription as monitored by cell-based reporter
assays. These results show that recognition of the middle of the
consensus motif is essential for PutA binding and proper
transcriptional control.
The basis for these results is evident from the crystal structure. The
mutated triplet corresponds to base pairs T9:A13, G10:C12, and C11:G11
of the structure. As shown in Fig. 5B, the protein directly
contacts T9, C12, C11 and G11. Mutation of T9 to C eliminates van der
Waals interactions between the thymine C5 methyl and Gly7(B) (Fig.
7c). Replacement of C12 with T eliminates the hydrogen bond with the
backbone carbonyl of Thr5(A) (Fig. 7b). In fact, this mutation
would position two hydrogen bond acceptors - carbonyl of Thr5(A) and O4
carbonyl of thymine - close to each other, which is unfavorable.
Moreover, the C5 methyl of the thymine would clash with Thr4(A).
Interestingly, changing C11:G11 to T:A is predicted to have little
effect on the protein-DNA interaction surface. Mutation of G11 to A
would preserve the hydrogen bond donated by Gly7(A) (Fig. 7b),
since both adenine and guanine have the hydrogen bond donor, N7.
Likewise, one can imagine the thymine O4 carbonyl engaging Thr5(B) in a
hydrogen bond analogous to the one formed with the C11 in Fig. 7B.
Thus, the observed deleterious effect on binding due to the GTcatA
triple mutation appears to be primarily due to the change of TG to CA.
Whereas E. coli has five exact repeats of the GTTGCA motif, Y. pestis
has single nucleotide substitutions in O1 (GTTaCA) and O2 (GTTGtA)
(Fig. S1). As described in the preceding paragraph, the G-to-A
variation in O1 is predicted to disrupt the protein-DNA interface. On
the other hand, the GTTGtA variation is likely to be accommodated by
the protein without significant structural penalty. Hence, the Y.
pestis O1 site may have a limited role in transcriptional control. We
note that Y. pestis has an additional GTTGCA sequence motif upstream of
O1, and that this site could substitute for a nonfunctional O1.
Importance of bases flanking the GTTGCA motif
Given the conservation of the GTTGCA motif in put intergenic regions,
it is not surprising that interactions with these bases are essential
for binding. Interestingly, the structure reveals that the footprint of
PutA52 extends beyond the GTTGCA motif, indicating that bases flanking
the consensus sequence may be important for operator recognition.
The structure shows that PutA52 contacts a 9-bp fragment, which we
denote XGTTGCAYZ. Lys9 interacts with XG and the complementary bases of
Y and Z. Based on the structure, it appears that G is preferred for X
and CC is preferred for YZ, because this sequence maximizes the number
of hydrogen bonds formed by Lys9 (eight total, four with each DNA
strand).
Operators 1-3 of E. coli have G for base X while operators 4 and 5 have
A (Fig. 1b). Substitution of A in place of G at position X can be
simulated by imagining adenine in place of G6 in Fig. 7a. This
change would eliminate one of the hydrogen bonds formed by Lys9. All
five operators have C for position Y except O4, which has A. The effect
of this variation can be seen by changing G7 in Fig. 7A to
thymine. Elimination of one hydrogen bond is again predicted. Operators
1 and 3 have T and A, respectively, at position Z, which requires Lys9
to interact with A and T, respectively. Both variations would eliminate
one hydrogen bond with Lys9, relative to optimal case of O2. This
simple hydrogen bond inventory analysis implies that PutA exhibits
different affinities for the five operator sites, with O2 predicted to
have the highest affinity (eight hydrogen bonds to Lys9) and O4 having
the lowest affinity (six hydrogen bonds).
This hypothesis was tested with ITC experiments that compared the
binding of PutA52 to two ligands differing only in the bases flanking
the consensus motif: O2 and O2fb4. The former ligand is the one used in
crystal structure determination. The latter is identical to O2 except
that it has the flanking bases of O4 (X,Y = A). These two ligands thus
mimic the two extremes of the predicted affinity spectrum of the PutA
operator sites.
The ITC analysis showed that PutA52 binds to O2 with fifteen times
higher affinity than O2fb4 (Table 2), in agreement with our
structure-based predictions. The binding enthalpy accounts almost
entirely for the difference in affinity (Table 2); ΔH for O2 is
more exothermic than that of O2fb4 by about 2 kcal/mol. That the
difference in affinity is enthalpic in origin is consistent with our
prediction that Lys9 forms more hydrogen bonds with the flanking bases
of O2 than O4. These results suggest that the five operator sites are
nonequivalent in terms of binding affinity, which is potentially
significant because differential binding could be important for proper
transcriptional regulation.
In light of these results, it is interesting that PutA is a weaker
repressor of the putP promoter than the putA promoter, yet the highest
affinity operator (O2) is involved in repression of putP whereas the
lowest affinity operator (O4) is involved in repression of putA. We
suggest that PutA-DNA binding affinity is only one of several factors
to consider when assessing the potential impact of the operators on
transcriptional repression. Other key factors include the number of
transcription start sites and the location of the operators relative to
the promoter regions. The putP gene was previously shown to have
multiple transcription start sites and three functional
promoters.26 The three putP promoters are positioned 14, 26, and
58 base pairs upstream of the O1 site. Thus, PutA binding at O1 and O2
is not predicted to directly interfere with RNA polymerase at each of
the putP promoters resulting in weaker repression of the putP gene by
PutA. On the other hand, the putA gene has only one promoter and a
single transcriptional start site with O3 and O4 located within the
putA promoter region. Thus, these sites are positioned optimally for
PutA to interfere with RNA polymerase, which could explain why PutA is
a stronger repressor of putA expression.
Parenthetically, this ITC analysis underscores the value of examining
binding reactions in buffers having distinct ionization enthalpies.
Besides revealing the involvement of protonation in protein-ligand
associations, inclusion of the buffer ionization enthalpy can, in
select cases, significantly improve the quality of the titration data.
For example, the intrinsic binding enthalpy for the interaction between
PutA52 and the O2fb4 oligo (0.18 kcal/mol) is effectively zero. Absent
the contribution of phosphate or Tris ionization, this binding reaction
would be invisible by ITC. Although the intrinsic enthalpy for the
PutA52-O2 association is somewhat larger (-1.79 kcal/mol), the
interaction would nonetheless be difficult to characterize in phosphate
buffer alone because the heat of buffer ionization reduces the observed
enthalpy to just -0.8 kcal/mol. In striking contrast, the highly
endothermic Tris ionization event renders the reaction much more
amenable to analysis and facilitates, via a global fitting strategy,
treatment of the data collected in phosphate buffer.
Possible roles of PutA-specific residues
PutA is a unique member of the RHH family of transcription factors.
With a polypeptide chain in excess of 1300 residues, PutA is the
largest protein known to contain an RHH domain. Furthermore, to our
knowledge, PutA is the only protein to have a flavin redox regulatory
domain coupled to an RHH domain.
PutA is also distinguished from other RHH proteins at the primary
sequence level.24 In particular, Gly7 and Pro29 are absolutely
conserved among PutAs, yet rarely found in other RHH domains.
We suggest three possible roles for Pro29. First, proline may
facilitate initiation of αB and thus help position Thr28 and His30 for
interaction with the DNA backbone. Second, Pro29 may provide a steric
“backstop” for the DNA backbone and thereby contribute to recognition
of a 9-nucleotide fragment of B-form DNA. A third possibility is that
Pro29 may be donating C-H…O hydrogen bonds to the DNA backbone. This
suggestion is based on the observation Pro29 C[δ] forms close contacts
with oxygen atoms of the phosphate backbone (Fig. 7a).
There is precedent for proline C[δ] donating hydrogen bonds. For
example, when proline is located in the middle or C-terminus of an
α-helix, C[δ] donates hydrogen bonds to the backbone carbonyl 3-5
residues preceding the proline.33 These unconventional hydrogen
bonds enable proline to appear in α-helices despite lacking a free N-H
group for classic i to i+4 hydrogen bonding. We note that the CH…O
distance of 3.4 Å observed in the PutA52/O2 complex is identical to the
average distance for proline intrahelix C-H…O hydrogen bonds.33
The unique location of Pro29 at the beginning of αB, juxtaposed to the
DNA backbone, also supports a hydrogen bonding role. Analysis of
RHH/DNA structures shows that there are two conserved hydrogen bonds
donated by the backbone of residues at the N-terminus of αB to the DNA
backbone.30 We observe only one of these conserved hydrogen
bonds, and it involves the backbone N-H of His30 (Fig. 7a). Since
proline does not have a free N-H group, the second conserved hydrogen
bond is missing. We suggest that the unconventional C-H…O hydrogen
bonds substitute for the missing conserved hydrogen bond.
Gly7 represents an interesting sequence variation for the RHH family.
Typically, this position of the β-sheet is occupied by a polar residue,
such as Thr or Asn, which forms hydrogen bonds with DNA bases. The
PutA52/O2 structure shows that Gly7 participates in base recognition,
despite lacking a side chain. It donates a hydrogen bond to a guanine
base and forms van der Waals contacts with the C5 methyl group of
thymine.
Gly7 may underlie a more global structural aspect of DNA recognition by
PutA: deep penetration into the major groove. Absence of a side chain
at this position allows the β-sheet to penetrate further into the major
groove, compared to other RHH proteins. As a measure of depth of
penetration, we calculated the distance of closest approach between the
DNA axis and each of the C[α] atoms of the three canonical β-sheet
residues responsible for base recognition (Thr5, Gly7, Lys9 in PutA).
The values for PutA52/O2 are 5.0 Å, 5.1 Å, and 8.2 Å for chain A and
7.3 Å, 6.0 Å, and 7.4 Å for chain B. The corresponding values for
Arc,34 MetJ,35 CopG,36 NikR,37 omega38
and FitA39 bound to DNA span the ranges 8.0 - 11.9 Å, 5.8 - 8.1
Å, and 8.2 - 11.7 Å, respectively, for these three residue positions.
Thus, PutA52 penetrates deeper into the major groove than the other RHH
proteins. The role of this close encounter in transcriptional
regulation of the put regulon is unknown, but the universal
conservation of Gly7 in PutAs, and its absence in the rest of the RHH
family, suggests functional significance.
Materials and Methods
Materials
Chemicals and buffers were purchased from Fisher Scientific and
Sigma-Aldrich, Inc. unless otherwise stated. Restriction endonucleases
and T4 DNA ligase were purchased from Fermentas and Invitrogen,
respectively. BCA reagents used for protein quantitation were obtained
from Pierce. Goat anti-rabbit secondary antibody was purchased from
Amersham Inc. E. coli strains XL-blue and BL21 DE3 pLysS were purchased
from Stratagene. E. coli strain JT31 putA- lacZ- was a generous gift
from J. Wood (University of Guelph, Guelph, ON, Canada). Synthetic
oligonucleotides for site-directed mutagenesis, cloning, DNA-binding
assays and co-crystallization were purchased from Integrated DNA
Technologies. All experiments used Nano-pure water. LB medium and
Terrific broth were used for general culture growth and protein
production, respectively, while M9 minimal medium was used for
cell-based transcription assays.
Gel-shift assays
Full-length PutA and PutA52 were expressed as C-terminally His-tagged
proteins from vector pET23b (Novagen) and purified as described
previously.23,24,40 The C-terminal His tags were
retained after purification. Purified full-length PutA was dialyzed
into 50 mM Tris (pH 7.5) containing 10 % glycerol and stored at -70 °C.
PutA52 was dialyzed into 50 mM phosphate buffer (pH 7.3) containing 200
mM NaCl and stored at -70 °C. The concentrations of the PutA proteins
were determined using the BCA method (Pierce) with bovine serum albumin
as the standard and spectrophotometrically using molar extinction
coefficients of 12,700 M-1 cm-1 at 451 nm for PutA and 6970 M-1
cm-1 at 280 nm for PutA52.24,41
Nondenaturing gel electrophoretic mobility shift assays were used to
test the binding of the PutA proteins to the put control intergenic DNA
as previously described.23,41 Different regions of the put
control DNA (putC) were PCR amplified (non-labeled) using synthetic
primers and purified. The purified products were incubated with
full-length PutA (0, 0.6, and 1.5 μM) for 20 min at 20 °C in 50 mM Tris
buffer (pH 7.5, 100 mM NaCl). The protein-DNA complexes were separated
using a native polyacrylamide gel (4 %) at 4 °C. The gel was then
stained with ethidium bromide and visualized by Bio-Rad Quantity One.
Binding assays with synthetic oligonucleotides corresponding to base
pairs 183-210 (O1), 342-365 (O3/4), and 388-412 (O5) of putC were
performed similarly. The concentration of oligonucleotide for these
assays was 100 nM. Duplex DNA of each oligonucleotide was prepared by
annealing the complementary oligonucleotides in buffer (10 mM Tris, pH
8.0, 50 mM NaCl, 1 mM EDTA) by first heating at 95 °C for 5 min and
then gradually cooling down the sample to room temperature.
Gel-shift assays utilizing fluorescently labeled put intergenic DNA
were also performed. The synthetic oligonucleotide (M13 forward primer)
was 5′ end-labeled with IRdye-700 (LI-COR, Inc.) and used as one of the
primers in a PCR reaction to amplify wild-type put intergenic DNA or
mutant put intergenic DNA containing different combinations of PutA
binding site mutations. The resulting IRdye-700 labeled put intergenic
DNA was purified and quantitated by measuring the nucleic acid
concentration at 260 nm and the absorbance of the IRdye-700 at 685 nm
using an extinction coefficient of 170 mM-1 cm-1 according to the
recommendations of the manufacturer. PutA (0-900 nM) or PutA52 (0-200
nM) was incubated with 2 nM put intergenic DNA in a total volume of 25
μl in 50 mM Tris, 50-250 mM NaCl, pH 7.5, containing 10 % glycerol for
20 min (20 °C) before electrophoresis. Calf thymus competitor DNA (100
μg/ml) was also added to the binding mixtures to prevent nonspecific
protein-DNA interactions. The PutA-DNA and PutA52-DNA complexes were
separated using a native polyacrylamide gel electrophoresis at 4 °C.
The gels were visualized using a LI-COR Odyssey Imager.
LacZ reporter assays
E. coli strain JT31 putA- lacZ- was cotransformed with PutA-pUC18 and
the reporter construct P[putA]:lacZ-pACYC184 or P[putP]:lacZ-pACYC184.
Details about the cloning procedures and the primers used for
generating the above constructs are provided in the supplemental
material and Table S1. To test which PutA binding sites are critical
for repressing put gene expression, E. coli strain JT31 putA- lacZ-
containing different combinations of the PutA-pUC18 construct and the
P[putA]:lacZ or P[putP]:lacZ reporter constructs were grown at 37 °C in
M9 minimal medium supplemented with ampicillin (50 μg/ml), kanamycin
(40 μg/ml) and chloramphenicol (34 μg/ml) to OD ~ 1.0. PutA
expression from the lac promoter on pUC18 was not induced as no
isopropyl-β-D-thiogalactopyranoside was added to the culture medium.
Cells from the various cultures were pelleted, resuspended in Tris-HCl
buffer (20 mM, pH 7.5) and broken using the B-PER II bacterial protein
extraction reagent from Pierce (20 mM Tris-HCl, pH 7.5).23
β-galactosidase activity assays were performed in a 1-ml volume of 100
mM sodium phosphate, pH 7.3, containing 1 mM MgCl, 50 mM
β-mercaptoethanol, and 2 mM o-nitrophenyl-β-D-galactopyranoside. The
initial velocity was determined by measuring the increase in absorbance
at 420 nm. The reported β-galactosidase activities are averaged values
from four independent experiments.
Expression of PutA was confirmed by Western blot analysis using an
antibody directed against a polypeptide containing PutA residues 1-47
(PutA47). PutA47 was purified without a 6xHis tag as described
previously.23 Antiserum directed against purified PutA47 was
prepared by Proteintech Inc. For Western blot analysis, cell pellets
from 5 ml culture grown in minimal medium (OD ~ 1.0) were resuspended
in 100 μL of SDS sample buffer and boiled for 10 min. After SDS
denaturing electrophoresis the protein bands were transferred onto a
sequi-blot polyvinylidene difluoride (PVDF) membrane (Bio-Rad, 0.2 μm
pore size) using an EBU-4000 semi-dry electrophoretic blotting system.
Immunoreactive bands were detected using Enhanced chemiluminescence
Western blotting reagents (Amersham Inc.).
Crystallization
Exhaustive crystallization experiments were conducted using two
different RHH domain constructs paired with several different DNA
fragments, as described in Supplemental Material. Successful
co-crystallization required the use of an RHH domain construct
consisting of E. coli PutA residues 1-52 (PutA52) fused to a cleavable
N-terminal histidine tag. Expression and purification protocols for
this protein have been described.24 The N-terminal tag was
removed by proteolysis prior to crystallization trials as
described.24 The purified, tag-free protein was concentrated
using an Amicon Ultra centrifugal filtration device (MWCO 5,000) to
10.8 mg/mL in a buffer of 20 mM Tris pH 8.0, 500 mM NaCl, 20 mM
imidazole. The protein concentration was estimated with the BCA method.
The oligonucleotide used for co-crystallization with PutA52 corresponds
to nucleotides 211-231 of the put control region, which contains the
second of five operator sites for PutA (denoted O2 in Fig. 1):

5′-TTTGCGGTTGCACCTTTCAAA-3′(strand 1)

3′-AAACGCCAACGTGGAAAGTTT-5′(strand 2)

Each DNA strand was dissolved in 10 mM Tris, 50 mM NaCl, 1 mM EDTA, pH
8.0 to a concentration of 6 mM. The two strands were annealed as
follows. Equal volumes of the oligonucleotide solutions were combined,
and the mixture was placed in a water bath at room temperature. The
temperature of the bath was then set to 94 °C, and once the target
temperature was reached, the power to the bath was turned off to slowly
cool the sample back to room temperature.
The PutA52 and dsDNA stock solutions were mixed so that the molar ratio
of PutA52 dimer to dsDNA was approximately 1:3. The mixture was
injected onto a Sephacryl S-100 HiPrep 16/60 gel filtration column
equilibrated with 10 mM Tris, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, pH
8.0. Fractions were pooled and concentrated to 11.6 mg/mL (BCA assay)
using an Amicon Ultra centrifugal filtration device (MWCO 10,000).
The PutA52/DNA mixture was input to several crystal screens to identify
initial crystallization conditions. The crystals used for data
collection were obtained directly from Index Screen reagent 54, which
consists of 30 % PEG-MME 550, 50 mM CaCl, and 100 mM Bis-Tris pH
6.5.
The crystals were prepared for low temperature data collection by
soaking them in a solution of 32 % PEG-MME 550, 50 mM CaCl, 100 mM
Bis-Tris pH 6.5, 15 % PEG 200. The crystals were then picked up with
Hampton loops and plunged into liquid nitrogen.
X-ray diffraction data collection, phasing and refinement
Several crystals were analyzed at Advanced Light Source beamline 4.2.2
using a NOIR-1 CCD detector. The data were integrated with
MOSFLM42 and scaled with SCALA.43 The crystals have space
group C2 with unit cell parameters of a = 90.9 Å, b = 44.1 Å, c = 55.2
Å, and β = 101.5°. The asymmetric unit contains one PutA52 dimer and
one DNA duplex, which corresponds to 50 % solvent and V[M] = 2.3
Å3/Da.44,45 The best data set had a high resolution limit
of 2.25 Å, and consisted of 180 frames collected with oscillation angle
of 1°/frame, exposure time of 7 s/frame and detector distance of 120
mm. Data collection and processing statistics are listed in Table
1.
Table 1
Table 1
Data Collection and Refinement Statisticsa
The structure was solved using molecular replacement with a PutA52
dimer serving as the search model. CNS was used for molecular
replacement calculations.46 The fast-direct method was used for
cross-rotation function calculations. Prior to translation function
calculations, the orientations from the cross-rotation function
calculation were optimized with Patterson correlation refinement using
two groups corresponding to the two protein chains of the dimer. The
top solution from the translation function calculation had correlation
coefficient of 0.257. Rigid body refinement resulted in a model with
R[cryst] = 0.488 and R[free] = 0.493 for data to 3.0 Å resolution.
Simulated annealing refinement in CNS lowered the R-factors to R[cryst]
= 0.450 and R[free] = 0.467 for all reflections.
The model from simulated annealing was used as the starting point for
iterative cycles of model building in COOT47 and refinement with
TLS in REFMAC5.48 After a few cycles, the R-factors for a
protein-only model were R[cryst] = 0.416 and R[free] = 0.427. At this
point, electron density representing DNA base pairs was evident. The
DNA part of the model was gradually built up over about two dozen
cycles of model building and refinement. Solvent was added during the
latter stages of model building.
The final model consists of one PutA52 dimer, one dsDNA molecule and 27
water molecules. The modeled protein chains include residues 3 - 46 for
chain A and 4 - 48 for chain B. The DNA strands include nucleotides 4 -
21 for strand 1 and 1 - 19 for strand 2. Electron density near the end
of the DNA duplex containing nucleotides 1-3 of strand 1 and 19 - 21
for strand 2 was rather weak, and indicated fraying of the base pairs
and possibly more than one conformation. But, the density was not of
sufficient quality to allow reliable modeling of this end of the DNA
ligand. We note that this end of the oligonucleotide is far from the
protein-DNA interface. Refinement statistics are listed in Table
1.
Structure analysis
Structures were analyzed graphically using COOT and PyMOL.49 CNS
was used to calculate buried surface area.46 DNA conformation was
analyzed with 3DNA.28 The depth of penetration of the β-sheet
into the DNA major groove was analyzed for RHH proteins. An operational
definition of penetration depth was adopted for this purpose as the
shortest distance between selected Cα atoms of the β-sheet and the DNA
helical axis. DNA helical axes were calculated using 3DNA.28 The
9 - 10 base pairs corresponding to the region contacted by the protein
were used for the axis calculation. Distances between C[α] atoms and
DNA axes were calculated using a program written by Damian Coventry,
which implements theory by Paul Bourke.
Isothermal titration calorimetry (ITC)
ITC experiments were conducted at 25 °C in a VP-ITC calorimeter
(MicroCal, LLC). Prior to analysis, the protein and oligonucleotide
were dialyzed extensively against the appropriate reaction buffer,
which was either 50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 8.0, or 50 mM
sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 8.0. The dimeric
quaternary structure of PutA52 was confirmed by equilibrium analytical
ultracentrifugation under conditions similar to those used in ITC
experiments (20 μM PutA52 in 50 mM Tris, 50 mM NaCl, pH 8.0). The data
could be fit very well to a single-species model with apparent
molecular weight corresponding to that of the homodimer (~13.5 kDa). No
evidence of a monomeric species was present at 20 μM. Sample and
titrant were degassed under vacuum immediately before being loaded into
the sample cell and buret, respectively. Following thermal
equilibration, aliquots (7 or 10 μL) of titrant were added to the 1.41
mL sample at 240-second intervals. A 2 μL pre-injection was included at
the start of each titration. The heat associated with this addition -
invariably inaccurate due to diffusion of titrant from the buret during
the equilibration period - was neglected during the fitting process.
Samples of PutA52 were titrated with two oligonucleotides, designated
O2 and O2fb4 (O2 with flanking bases of O4). O2 is the oligonucleotide
used for co-crystallization. O2fb4 is identical to O2 except that the
base pairs flanking the consensus motif are those of operator 4
(Fig. 1B):

5′-TTTGCAGTTGCAACTTTCAAA-3′(strand 1)

3′-AAACGTCAACGTTGAAAGTTT-5′(strand 2)

Experiments were conducted in both phosphate and Tris, two buffers with
distinct ionization enthalpies. The raw data were integrated with
software supplied with the instrument. Blank titrations, injection of
titrant into buffer, were performed for each oligonucleotide-buffer
combination. The average injection heats associated with these
experiments were used to correct the corresponding protein titrations
for the nonspecific heat of mixing/dilution.
The apparent protein-DNA binding enthalpies differed profoundly in Tris
and phosphate, indicating that the PutA52-DNA interaction is
accompanied by protonation. Accordingly, the data from the two buffer
systems were subjected to simultaneous least-squares analysis,
employing a model that explicitly includes the heat of buffer
ionization. The following equation describes the cumulative heat after
the ith titrant addition:
equation M1
(1)
where V is the sample cell volume, [M][t] is the total protein
concentration, ΔH is the intrinsic binding enthalpy, ΔH[buf] is the
heat of buffer ionization, n is the number of protons taken up by the
protein-DNA complex during the binding reaction, K[ap] is the apparent
association constant, and [DNA] is the concentration of free DNA.
Because protein-DNA binding is linked to protonation, the apparent free
energy change for the reaction (ΔG[ap]) includes a contribution from
buffer ionization (ΔG[buf]):
equation M2
(2)
Thus,
equation M3
(3)
or
equation M4
(4)
where K is the intrinsic binding constant for the protein-DNA
association, and K[buf] is equal to
equation M5
(5)
In equation 5, [BH+] and [B] represent the concentrations of the
conjugate acid and base forms of the reaction buffer, and pK[a] is the
appropriate value for the particular buffer under consideration. K, ΔH,
and n were global fitting parameters; pH was a fixed global parameter;
and ΔH[buf] and pK[a] were fixed titration-specific parameters obtained
from the literature.50
The ith injection heat (q[i]) was modeled as the difference in the
cumulative heats associated with the ith and (i+1)th additions:
equation M6
(6)
The second term in equation 6 is a correction for the heat
associated with the volume of solution displaced from the sample cell
by the ith titrant addition, where dV[i] is the volume of the ith
injection. Fitting was performed in Origin (v. 7.5, OriginLab),
employing a LabTalk script generated in-house.
PDB accession code
Atomic coordinates and structure factor amplitudes have been deposited
in the PDB51 as entry 2RBF.
Supplementary Material
supplementary
Click here to view.(566K, pdf)
Acknowledgments
This research was supported by NIH grants GM065546 (JJT) and GM061068
(DFB), and NSF grant MCB0091664 (DFB). CAB was supported by a
postdoctoral fellowship from the National Library of Medicine
(2-T15-LM07089-14). We thank Damian Coventry and Paul Bourke for
providing computer code that was used in penetration depth
calculations. Part of this research was performed at the Advanced Light
Source, which is supported by the Director, Office of Science, Office
of Basic Energy Sciences, Materials Sciences Division, of the U.S.
Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence
Berkeley National Laboratory. This work is a contribution of the
University of Nebraska Agricultural Research Division, supported in
part by funds provided through the Hatch Act. This publication was also
made possible by NIH Grant Number P20 RR-017675-02 from the National
Center for Research Resources. Its contents are solely the
responsibility of the authors and do not necessarily represent the
official views of the NIH.
Abbreviations used
PRODH  proline dehydrogenase
P5CDH  Δ1-pyrroline-5-carboxylate dehydrogenase
PutA   Proline utilization A
PutA52 polypeptide corresponding to residues 1-52 of E. coli PutA
RHH    ribbon-helix-helix
PutA47 polypeptide corresponding to residues 1-47 of E. coli PutA
bp     base pair
ITC    isothermal titration calorimetry